A total of 305 stools samples during the period of October 2011 to June 2012 were collected from patients attending Brack General Hospital and Medical Technology, Department of Brack, Al-Shati, Libya. All stool samples were examined by direct smear preparation in normal saline, iodine, and eosin stains and 4 concentration methods (formalin-ether, normal saline sedimentation, zinc sulfate, and Sheather's sugar flotation). Of the 305 samples, 18.03% stools were found positive for protozoan parasites in direct smear microscopy. Normal saline sedimentation and zinc sulfate flotation detected 27.21% and 23.6% positive samples, respectively. However, formalin-ether was found to be the most sensitive method. Sheather's sugar flotation failed to detect Entamoeba histolytica/dispar, Entamoeba coli, and Blastocystis hominis. B. hominis was the most common parasite among the patients. Concentration techniques showed significantly higher (P < 0.05) detection rates of parasites compared with direct smear microscopy.

Generally, the prevalence rate of gastrointestinal parasites varies from one area to another depending on the degree of personal and community hygiene, sanitation, and climatic factors [1],[2] and different diagnostic techniques may also influence their detection. [3] Young children and adolescents in developing countries display highest prevalence of intestinal parasites and burden of morbidity. Intestinal parasites are linked to diarrhea, dysentery, weight loss, malnutrition, anemia, abdominal pain, and other gastrointestinal ailments. [4] Also chronic parasitism impairs physical development and cognitive functions of growing children. [5] Gastrointestinal parasites can cause infection in both humans and animals. Some of these are potentially zoonotic. [6],[7] Moreover, intestinal opportunistic parasitic infections are also a serious public health problem, which has been increasing in recent years in developing countries. [8],[9]

Relatively, little work has been done on the diagnosis of intestinal parasites in Libya. [10],[11],[12],[13],[14] and complete information of intestinal parasites is lacking. The most common intestinal parasites in Libya are Blastocystic hominis followed by Entamoeba histolyitca/dispar, Giardia lamblia, and Entamoeba coli.[11],[15],[16] Moreover, Cryptosporidium spp. infections have been reported in Libya among patients, particularly in children with diarrhea. [14],[17],[18],[19] Relatively, low infection rates of helminthes have also been reported in this country. [11],[14]

The diagnosis of intestinal parasites is performed by microscopic examination of stools, which has been recognized as the "gold standard" for a long time. [20] Stool examination is regularly used for parasitological diagnosis; however, this system of diagnosis has some limitations, such as lack of sensitivity, especially when the diagnosis relies on a single microscopic examination of stool specimens. [21] This is probably due to irregular or intermittent shedding of parasites or the low numbers of their excretion in feces. The most commonly used methods for detecting intestinal parasites from stool examination include direct smear microscopy and concentration techniques. [22] However, recent reports have indicated that concentration techniques showed variable efficiency in the detection of intestinal parasites in stools. [22],[23],[24] Therefore, multiple stool examinations or more than one copro-parasitological method should be used for diagnosis of intestinal parasites, which are usually undetected or at least unrecognized during routine direct smear microscopy in laboratories. So far, different parasitological methods have not been evaluated in Libya for detection of intestinal parasites in stool samples. Thus, studies are required for evaluation of different parasitological methods for the diagnosis of intestinal parasites in Libya to find out whether these methods would increase the detection capacity of parasites in stools or any variations if present. Therefore, the aim of present study was to compare direct smear microscopy and 4 concentration methods: Normal saline sedimentation, formalin-ether sedimentation, zinc sulfate flotation, and Sheather's sugar flotation technique for the detection of intestinal protozoa in clinical stool specimens.

Materials and Methods

This cross-sectional study was conducted during the period of October 2011 to July 2012. A total of 305 stool samples were collected from patients attending Brack Hospital and Medical Technology Laboratory, Brack for the detection of intestinal parasitic infection. Out of 305, 107 were males and 198 were females. All patients who were able to provide stool samples during their visits were included in the study. Necessary permissions to conduct this study were obtained from the authorities of Brack Hospital and Department of Medical Technology Laboratory, Brack before commencement of the study. Each sample was processed and examined immediately after collection, by routine direct fecal smear microscopy and concentration techniques using normal saline and Lugol's iodine preparations to record the prevalence of intestinal parasites in patients. Direct wet mount was used primarily to detect motile forms of protozoa, allowing study of the motility of the organisms, which are often characteristic. Formalin-ether sedimentation concentration technique was used for the concentration of intestinal parasites as described by Cook, and Chessbrough. [25],[26] Simple normal saline sedimentation technique was carried out. [27]

Concentration of stool specimens was accomplished by zinc sulfate floatation method [28] to demonstrate cysts and eggs of intestinal parasites. Sheather's sugar floatation, sugar flotation method was used for concentration of intestinal parasites in the stools, as described by Anderson. [29] Modified Ziehl-Neelsen (ZN) acid-fast stain was used to identify the oocysts of Cryptosporidium spp. in the fecal specimens following the procedures described by Garcia et al.,[30] and Baxby et al.[31]

Statistical analysis and chi square test were carried out by using SPSS version 16. A probability (P) value of less than 0.05 was considered as significant whenever appropriate.

B. hominis was the most common parasite among the patients. Sixty-three (20.65%) samples were positive upon use of concentration methods along with direct smear microscopy, while 51 (16.72%) showed positive detection when direct smear microscopy alone was used. About 9.50%, 0.65%, and 1.31% of the samples were found to be positive for E. histolytica/dispar, E. coli, and G. lamblia, respectively."

None of 305 stool specimens was found positive for helminthes eggs or larvae. Cryptosporidium oocysts were detected only in 4 and 7 stool specimens by formalin-ether sedimentation and Sheather's sugar flotation procedures, respectively. Single parasitic infections had a higher prevalence (96.60%) compared to multiple infections (3.37%) protozoa among patients.

Discussion

In Libya, diagnosis of intestinal parasitic infections in clinical laboratories is made by routine direct smear microscopy of stools in normal saline or iodine preparations. Evaluation of different concentration procedures has not been done for clinical specimens. Therefore, data are not available for comparison. The present study describes comparison of 4 concentration techniques for the detection of parasites in stools and makes data available for comparison.

The results of this study demonstrates the value of using both sedimentation and flotation procedures. Formalin-ether sedimentation confirmed an increase of detection efficiency of parasites in stools compared to other concentration methods used in the study. This observation agrees favorably with other similar studies, [22],[32],[33],[34] which reported superiority of formalin-ether over other concentration procedures.

The results of this study revealed the distinctive superiority of concentration techniques over direct smear microscopy (P < 0.05). This result agrees with other studies, [14],[22],[35],[34],[35],[36],[37],[38] which clearly indicated that formalin-ether sedimentations are advantageous over direct smear preparations.

In the present study, results obtained with sedimentation procedures (formalin-ether and normal saline) and flotation methods (zinc sulfate) were almost similar for the detection of protozoa cysts (E. histolytica/dispar, E. coli, G. lamblia, and B. hominis). Similar findings have been observed by Truant et al.,[39] who reported no significant difference between sedimentation (formalin-ether and formalin-ethyl acetate) and flotation (zinc sulfate) procedures for the identification of protozoan cysts. However, Bartlett et al.[40] and Truant et al., [39] reported that compared to zinc sulfate method, formalin-ether method missed detection of some protozoan cysts. Moreover, Methanitikorn et al.,[41] also reported that formalin-ether sedimentation detected a higher percentage of positive samples of protozoa than formalin-Tween concentration technique. However, Bartlett et al., [40] and Truant et al.,[39] also observed that compared to zinc sulfate method, formalin-ether method missed detection of some protozoan cysts. Sheather's sugar flotation method in the present study, detected significantly lower percentage of positive samples. This could be due to disintegration of certain parasites in Sheather's sugar solution as its specific gravity is high. This result agrees with similar observation reported by Scott et al.,[36] who observed that many parasites were detected by formalin-ether method but were not seen in Sheather's sugar flotation of parasites in stool specimens.

This study also demonstrated that among patients in Wadi Al-Shati Province, there is 29.18% incidence of intestinal protozoa. This finding is within the range of prevalence rate reported from other cities of Libya. [11],[14] However, results differ from El-Buni and Khan, [42] Sadaqa and Kassem, [12] and Ben-Mousa, [13] who reported lower prevalence rates of intestinal parasites among Libyan population.

In the present study, B. hominis was the most common parasite reported in the stool specimens of patients. Similarly, studies from other regions of Libya [11],[12],[14],[15],[43] have reported a prevalence rate of B. hominis range between 18.5% and 29.5%. With particular attention to B. hominis, sedimentation (formalin-ether and normal saline) and flotation (zinc sulfate) techniques were more effective in detecting of this organism. A similar result was reported by Truant et al.,[39] who found that formalin-ether was suitable for the detection of B. hominis.

In Libya, limited studies have been carried out for the diagnosis of cryptosporidiosis. [17],[18],[19],[44] These studies reported an incidence of cryptosporidiosis varies from 3.19% to 13.0%. Regarding cryptosporidial infection in the present study, 305 stool specimens were concentrated for parasites by using formalin-ether sedimentation and Sheather's sugar flotation methods for the detection of oocysts of Cryptosporidium spp. Only 4 (1.31%) and 7 (3.60%) samples were found positive for Cryptosporidium spp. by formalin-ether and Sheather's sugar flotation techniques, respectively. There was no significant difference (P > 0.05) between two methods in detecting overall infection of this organism. Almost similar incidence of cryptosporidiosis (2.51%) has been reported by Gelani et al., [14] among random population of Wadi Al-Shati Province. Scott et al., [36] compared formalin-ether and Sheather's sugar flotation procedures for the detection of oocysts of Cryptosporidium spp. and found that Sheather's sugar flotation method detected slightly more number of positive samples compared to formalin-ether method. However, this difference was not statistically significant.

In the present study, direct smear microscopy missed the detection of parasites in stool specimens. Both formalin-ether and normal saline sedimentation techniques appear to be suitable and emphasized to achieve the maximum detection of parasites in clinical stool samples. However, zinc sulfate has advantage. Wet mounts prepared from the surface films have less background fecal detritus than formalin-ether or normal saline sedimentation wet mounts. This would be helpful for individuals with limited experience in microscopic screening.

Conclusion

This study shows that the prevalence of intestinal protozoan parasites is relatively high in the studied population. This study also indicated that formalin-ether procedure provides a suitable alternative to normal sedimentation and zinc sulfate flotation method. Formalin-ether method is recommended as a single concentration method for the routine examination of the intestinal parasites in clinical stool samples.